U.S. patent number 3,627,688 [Application Number 04/786,431] was granted by the patent office on 1971-12-14 for stabilized aqueous enzyme containing compositions.
This patent grant is currently assigned to The Procter & Gamble Company. Invention is credited to Jim S. Berry, Charles B. McCarty.
United States Patent |
3,627,688 |
McCarty , et al. |
December 14, 1971 |
**Please see images for:
( Certificate of Correction ) ** |
STABILIZED AQUEOUS ENZYME CONTAINING COMPOSITIONS
Abstract
Stabilized aqueous enzyme compositions containing a protease
and/or an .alpha.-amylase and a stabilizing agent selected from the
group consisting of dialkyl glycol ethers; heterocyclic oxyethers;
and dialkyl ketones are described. These compositions, which can
also contain a nonionic or zwitterionic detergent, are useful
cleaning compositions particularly in the removal of soils and
stains from textile materials. FIELD OF THE INVENTION This
invention relates to stabilized proteolytic and/or amylolytic
enzyme-containing compositions useful in the removal of stains and
soils from textile materials. More particularly, it relates to
aqueous enzyme-containing compositions containing a minor amount of
a stabilizing agent which preserves the enzymatic activity of the
proteolytic and/or amylolytic enzyme. The use of proteolytic and
amylolytic enzymes in laundry products is known. See for example,
U.S. Pat. No. 1,882,270 to Frelinghusen Oct. 11, 1932) and Jaag,
"Effect of Enzymatic Detergents," Seifen Ole, Fette, Wachse, 88,
No. 24, pp. 789-793 (Nov. 1962). These enzymes aid in the laundry
process by attacking soil and stains found on soiled fabrics and
decomposing and/or altering them so as to render them more
removable during laundering. Enzymatic materials suitable for
laundering are expensive and powerful materials which must be
judiciously formulated and used. These enzymes when employed in
aqueous compositions are unstable and suffer appreciable
destruction, particularly during long periods of storage, resulting
in substantial loss of soil- and stain-removing efficacy. The loss
in enzymatic activity is particularly severe under conditions of
high temperature. Moreover, such aqueous solutions often contain
additional components which are desirable from the standpoint of
detergency and cleaning but which exert a harmful effect on the
enzymatic material. The result is further loss of enzymatic
activity. Numerous attempts have been made in the art to provide
aqueous enzyme-containing compositions wherein the enzymatic
activity is preserved by the incorporation of stabilizing agents.
U.S. Pat. Nos. 3,050,445 (Aug. 21, 1962), 3,095,358 (June 25, 1963)
and 3,325,364 (June 13, 1964) illustrate attempts to stabilize
aqueous solutions of enzymes. None of the prior art methods of
stabilizing aqueous enzyme-containing compositions against loss in
enzymatic activity have been entirely satisfactory. Generally,
these methods provide aqueous compositions which either lose
appreciable enzymatic activity upon long periods of storage or
require the employment of large amounts of stabilizing agents to
effect suitable levels of enzyme stabilization. It is therefore an
object of this invention to provide stabilized aqueous solutions
which retain substantially their enzyme activity upon storage. It
is another object of this invention to provide aqueous proteolytic
and/or amylolytic enzyme-containing compositions stabilized
substantially against loss of activity by the presence of minor
amounts of an enzyme-stabilizing compound. Other objects of this
invention will be obvious from consideration of the invention which
is more fully described hereinafter.
Inventors: |
McCarty; Charles B.
(Cincinnati, OH), Berry; Jim S. (Springfield Township,
Hamilton County, OH) |
Assignee: |
The Procter & Gamble
Company (Cincinnati, OH)
|
Family
ID: |
19931441 |
Appl.
No.: |
04/786,431 |
Filed: |
December 23, 1968 |
Foreign Application Priority Data
Current U.S.
Class: |
510/284; 435/188;
435/263; 510/281; 510/494; 510/506; 510/530; 510/321 |
Current CPC
Class: |
C11D
3/38663 (20130101); C12N 9/96 (20130101) |
Current International
Class: |
C12N
9/96 (20060101); C11D 3/386 (20060101); C11D
3/38 (20060101); C11d 001/72 (); C11d 001/74 () |
Field of
Search: |
;195/63,68 ;424/94
;252/DIG.12,89,132,135,152,403,398,153,DIG.14 ;260/614,615 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Nature, Vol. 215, pp. 417-419, (1967). .
Yasumatsu et al., Agr. Biol. Chem., Vol. 29(7), pp. 665-71,
(1965)..
|
Primary Examiner: Rosdol; Leon D.
Assistant Examiner: Rady; Arnold I.
Claims
What is claimed is:
1. A stabilized aqueous enzyme composition consisting essentially
of by weight of the composition:
1. from 65 to 97 percent water;
2. from about 0.001 to about 1.0 percent enzyme selected from the
group consisting of proteases, .alpha.-amylases and mixtures
thereof;
3. from about 2 to about 27 percent of a stabilizing agent selected
from the group consisting of dialkyl glycol ethers having the
general formula
R.sub.1 O[CH.sub.2 CH.sub.2 O].sub.x R.sub.2 wherein R.sub.1 and
R.sub.2 are each alkyl of one to four carbon atoms and x is 1 to
10.
4. from zero to about 15 percent of a detergent selected from the
group consisting of nonionic detergents and zwitterionic
detergents.
2. The composition of claim 1 wherein the enzyme is an alkaline
protease.
3. The composition of claim 2 wherein the alkaline protease is
derived from Bacillus subtilis.
4. The composition of claim 1 wherein the enzyme is an
.alpha.-amylase.
5. The composition of claim 1 wherein the enzyme is a mixture of
protease and .alpha.-amylase.
6. The composition of claim 3 wherein the stabilizing agent is
present in an amount of about 5 to about 20 percent.
7. The composition of claim 6 wherein the stabilizing agent is
selected from the group consisting of ethylene glycol dimethyl
ether and diethylene glycol dimethyl ether.
8. The composition of claim 7 wherein from about 4 to about 10
percent of a nonionic or zwitterionic detergent is present.
9. The composition of claim 8 wherein the detergent is selected
from the group consisting of condensation products of 1 mole of
aliphatic alcohol having from eight to 22 carbon atoms with from 5
to 40 moles of ethylene oxide; 3-(N,N-dimethyl-N-alkylammonio)
propane-1-sulfonate wherein the alkyl has from eight to 22 carbon
atoms; and
3-(N,N-dimethyl-N-alkylammonio)-2-hydroxypropane-1-sulfonate
wherein the alkyl has from eight to 22 carbon atoms.
10. The composition of claim 9 wherein the pH is from about 5.0 to
about 10.0 and wherein the enzyme is present in an amount of about
0.01 to about 0.5 percent.
11. The composition of claim 10 wherein the water is present in an
amount of about 72 to about 95 percent.
Description
SUMMARY OF THE INVENTION
These and other objects of the present invention are achieved by
the provision of aqueous proteolytic and/or amylolytic
enzyme-containing compositions characterized by extended periods of
stabilization and containing minor amounts of certain organic
stabilizing agents. The aqueous compositions of the present
invention can additionally contain nonionic or zwitterionic
detergent components to enhance the stability of the enzymes in the
aqueous compositions of this invention and to provide the
stabilized compositions with excellent detergent properties. The
present invention is based in part on the discovery that dialkyl
glycol ethers, heterocyclic ethers, and dialkyl ketones, provide
enzyme stabilization for extended periods of time.
The stabilized aqueous enzyme-containing compositions of this
invention comprise:
1. from about 65 to about 97 percent water;
2. from about 0.001 to about 1.0 percent enzyme selected from the
group consisting of proteases, .alpha.-amylases, and mixtures
thereof;
3. from about 2 to about 27 percent of a stabilizing agent selected
from the group consisting of dialkyl glycol ethers having the
general formula
R.sub.1 O[CH.sub.2 CH.sub.2 O].sub.x R.sub.2
wherein R.sub.1 and R.sub.2 are each alkyl of one to about four
carbon atoms and x is one to about 10; heterocyclic oxyethers; and
dialkyl ketones of the general formula
wherein R.sub.1 and R.sub.2 are each alkyl of one to about four
carbon atoms; and
4. from zero to about 15 percent of a detergent selected from the
group consisting of nonionic detergents and zwitterionic
detergents.
The proteolytic enzymes which can be stabilized in aqueous solution
by the action of the hereinbefore described organic stabilizing
agents include the alkaline proteases, neutral proteases, and acid
proteases. Proteases in these classifications are generally derived
from fungal and bacterial sources. Enzymes derived from plant and
animal sources can also be utilized herein but are not as readily
classified in the above alkaline, neutral, and acid subclasses.
These enzymes are active in the pH range of from about 3 to about
11 and at temperature ranging from about 40 to about 170.degree. F.
Optimum activity of these proteases is generally exhibited in the
pH range of from about 5.0 to 10 and preferably from 6.0 to
9.5.
The proteases are particularly effective in degrading protein soil.
The proteases catalyze the hydrolysis of the peptide linkage of
proteins, polypeptides, and related compounds. Free amino and
carboxy groups are thus obtained and the long chain protein
structures are reduced to several shorter chains. These shorter
chains can easily be removed from their environment with water of
aqueous detergent compositions.
The alkaline proteases are particularly preferred enzymes for use
herein. Alkaline proteases which are suitable for use in this
invention include subtilisin, BPN', elastase, keratinase,
carboxypeptidase, amino peptidase, aspergillopeptidase A and
aspergillopeptidase B. Subtilisin and BPN' are especially preferred
for use herein. The alkaline proteases are particularly preferred
for use in this invention as they show optimum activity in the pH
range of normal detergent components, i.e., 7.5 to 10.5, and the
alkaline proteases show surprising stability in the compositions of
this invention.
The neutral proteases which can also be utilized in the
compositions of this invention include collagenase, chymotrypsin,
and trypsin and those proteolytic enzymes isolated from
Streptomyces species. Both chymotrypsin and trypsin show optimum
activity in the neutral to alkaline range.
Examples of acid proteases suitable for use herein include pepsin,
papain, and bromelin. Both papain and bromelin show optimum
activity in the acid to neutral range.
The .alpha.-amylases are also stabilized in the compositions of
this invention. All of the .alpha.-amylases show optimum activity
in the acid range. The .alpha.-amylases are particularly well
suited for breaking down starch molecules as they attack the
.alpha.-1,4-glycosidic linkages in starch. The remaining shorter
chains are easily removed from their environment with water or
aqueous solutions of detergents. The .alpha.-amylases may be
obtained from animal sources, cereal grains, bacterial or fungal
sources.
Commercial enzyme compositions containing the above-described
enzymes are suitable for use herein. These commercial enzyme
compositions are generally sold in a dry powdered form and are
comprised of from about 2 to about 80 percent active enzymes in
combination with an inert powdered vehicle such as sodium or
calcium sulfate or sodium chloride as the remaining 20 to 98
percent. The active enzyme content of commercial enzyme
compositions is the result of manufacturing methods employed and is
not critical herein so long as the finished compositions of this
invention have the specified enzyme content. The insoluble inert
materials are generally removed from the compositions of this
invention to provide compositions having suitable clarity and which
are free of precipitates.
Specific examples of commercial enzyme compositions suitable for
use herein and the manufacturer thereof include: Alcalase, Novo
Industri, Copenhagen, Denmark; Maxatase, Koninklijke Nederlandsche
Gist-En Spiritusfabriek N.V., Delft, Netherlands; Protease B-400
and Protease AP, Schweizerische Ferment A.G., Basel, Switzerland;
CRD-Protease, Monsanto Company, St. Louis, Mo.; Viokase, VioBin
Corporation, Monticello, Ill.; Pronase-P, Pronase-E, Pronase-AS and
Pronase-AF all of which are manufactured by Kaken Chemical Company,
Japan; Bioprase, Nagase & Co. Ltd., Osaka, Japan; Rapidase
P-2000, Rapidase, Seclin, France; Takamine, Bromelain 1:10, HI
proteolytic enzyme 200, Enzyme L-W and .alpha.-amylase, Miles
Chemical Company, Elkhart, Ind.; Rhozyme P-11 concentrate,
Pectinol, Rhozyme PF, Rhozyme J-25, Rohm & Haas, Philadelphia,
Pa. (Rhozyme PF and J-25 have salt and cornstarch vehicles and are
proteases having diastase activity); Amprozyme 200, Jacques Wolf
& Company, a subsidiary of Nopco Chemical Company, Newark, N.J.
and Wallerstein 627P, Wallerstein Company, Staten Island, N.Y.
CRD-Protease (also known as Monsanto DA-10) is a useful powdered
enzyme composition. CRD-Protease is reported to be obtained by
mutation of a Bacillus subtilis organism. It is comprised of
neutral and alkaline proteases and .alpha.-amylase. The neutral
protease has a molecular weight of about 44,000 and contains from
one to two atoms of zinc per molecule. The CRD-Protease can be used
in aqueous systems such as the present invention. The active enzyme
content of CRD-Protease on a weight percent basis generally ranges
from about 20 to about 75 percent.
Pronase-P, Pronase-E, Pronase-AS and Pronase-AF are powdered enzyme
compositions which can also be used to advantage in this invention.
These enzymes are produced from the culture broth of Streptomyces
griseus used for streptomycin manufacture. They are isolated by a
successive resin column treatment. The major component of Pronase
is a neutral protease, Streptomyces griseus protease. This enzyme
composition contains a calcium stabilizer salt and is fairly stable
over a wide pH range, e.g., 4 to 10; and is fairly stable over a
temperature range of 50.degree. to 150.degree. F.
Another enzyme composition preferred for use in the compositions of
this invention is Alcalase which is manufactured and sold by Novo
Industri A/S, Copenhagen, Denmark. Alcalase is described, in a
trade bulletin bearing that name which is published by Novo
Industry A/S, as a proteolytic enzyme preparation manufactured by
submerged fermentation of a special strain of Bacillus subtilis.
The primary enzyme component of Alcalase is subtilisin. In addition
to Proteases, Alcalase contains small amounts of .alpha.-amylase.
Alcalase is a fine grayish free-flowing powder having a crystalline
active enzyme content of about 6 percent. The remainder of the
powder having a crystalline active enzyme content of about 6
percent. The remainder of the powder is comprised primarily of
sodium sulfate, calcium sulfate and various inert organic vehicle
materials. Alcalase has unusually stable properties in the aqueous
compositions of this invention.
Biophase is a powdered enzyme composition which contains alkaline
proteases (BPN') and .alpha.-amylases. This enzyme composition can
be obtained with or without the presence of diluents such as sodium
and calcium sulfate.
Large variations in the amount of enzymes in the compositions of
this invention are contemplated. The compositions can contain from
about 0.001 to about 1 percent enzyme by weight of the composition.
For best results, the compositions preferably contain from 0.01 to
about 0.5 percent enzymes by weight. When one of the preferred
enzyme compositions is utilized herein, the compositions of this
invention preferably contain from about 0.1 to about 4.0 percent of
the enzyme composition as it is sold in commercial form, e.g., from
about 2 to about 80 percent active enzyme. The active enzyme
content of the aqueous enzyme composition of this invention should,
in any event, range between 0.001 and 1 percent as above
delineated.
The stabilizing agents which stabilize the enzymes described above
are water-soluble organic compounds selected from the group
consisting of dialkyl glycol ethers, heterocyclic oxyethers and
dialkyl ketones.
Examples of dialkyl glycol ethers of the formula
R.sub.1 O[CH.sub.2 CH.sub.2 O].sub.x R.sub.2
wherein R.sub.1, R.sub.2, and x have the same definitions
hereinbefore described include ethylene glycol dimethyl ether,
ethylene glycol diethyl ether, ethylene glycol di-n-propyl ether,
diethylene glycol dimethyl ether, triethylene glycol dimethyl
ether, the methyl ether of tetraoxyethylenated methanol, the ethyl
ether of pentaoxyethylenated ethanol, the methyl ether of
hexaoxyethylenated butanol, the butyl ether of decaoxyethylenated
butanol and the like. Preferred herein are the alkyl ethers of
ethylene glycol, diethylene glycol and triethylene glycol, i.e.,
those wherein x in the hereinbefore described formula is from 1 to
3. These compounds are preferred from the standpoint of enzyme
stabilization and ready availability.
Suitable cyclic ethers include the 5- and 6-membered oxyethers.
These include the water-soluble solvent ethers such as furan,
tetrahydrofuran, dioxane, pyran, and the like. Substituted
derivatives, e.g., C.sub.1 to C.sub.10 alkyl or alkoxy-substituted
or fused-ring cyclic ethers, can also be employed so long as the
compounds are water soluble and do not denature or otherwise injure
the enzyme. A preferred ether herein is tetrahydrofuran.
Examples of suitable ketones are acetone, methyl ethyl, ketone,
diethyl ketone, di-N-propyl ketone, diisopropyl ketone and the
like. It will be appreciated that the hereinbefore described
stabilizing agents can be employed singly or in combination.
The enzyme stabilizing compounds described above can be employed in
the compositions of this invention in effective amounts ranging
from about 2 to 27 percent by weight of the composition. The
precise amount employed will depend in part on the solubility of
the organic stabilizing compound, the nature of the enzyme
employed, the desired level of stabilization and economic
considerations. Preferably the stabilized compositions of the
invention are prepared to contain from about 5 to about 20 percent
of the stabilizing agent, the latter range being preferred from the
standpoint of optimum stabilizing effects for most enzymes,
particularly over long storage periods at high temperature.
It will be appreciated that variation in the levels of
stabilization herein will depend as well on the nature of the
proteolytic or amylolytic enzyme employed and the particular
stabilizing agent employed. When an alkaline protease of bacterial
origin is utilized in the aqueous compositions of the invention,
best results are obtained when the organic stabilizing agent is
ethylene glycol dimethyl ether (1,2-dimethoxyethane), diethylene
glycol dimethyl ether, acetone or tetrahydrofuran, these compounds
being preferred herein. These stabilizing agents employed in an
amount of about 10percent of the stabilized compositions of the
invention are effective in the preservation of a substantial
percentage of initial enzymatic activity. Table I, for example,
illustrates the preservation of at least about half of initial
activity after eight weeks of storage at 100.degree. F.
Preferred compositions herein are those containing mixtures of
proteases and .alpha.-amylases, these being preferred by reason of
their application in the removal of a wide variety of stains of
proteinaceous and starchy origin. These compositions preferably
contain a mixture of organic stabilizers adapted to the
stabilization of the particular enzymes employed. The stabilized
compositions of the invention can additionally contain other enzyme
stabilizing compounds, such as, alkanols, alkoxy-substituted
alkanols, polyhydric alcohols, e.g., ethylene glycol, sorbitol or
mannitol, gelatins and inorganic calcium or magnesium salts to
provide additional enzyme-stabilizing effects.
The stabilizing agents employed herein, in addition to providing
enzyme stabilization, aid the cleaning process insofar as they
possess solvent action. Preferred stabilizers from this standpoint
include ethylene glycol dimethyl ether, tetrahydrofuran and
acetone.
Water-soluble nonionic and zwitterionic detergents can be employed,
as optional ingredients, in the compositions of this invention.
These detergents enhance the storage stability of the enzymes
employed herein and significantly improve the detergent
characteristics of the compositions. Because of these useful
characteristics, it is preferred to include nonionic and
zwitterionic detergents in the aqueous enzyme compositions of this
invention. The nonionics and zwitterionics can be utilized herein
in amounts ranging from zero to about 15 percent, preferably from 4
to 10 percent, by weight of the enzyme compositions.
Examples of suitable nonionics for use herein includes:
1. The polyethylene oxide condensates of alkyl phenols, e.g., the
condensation products of alkyl phenols having an alkyl group
containing from about six to 12 carbon atoms in either straight
chain or branched-chain configuration with ethylene oxide, the said
ethylene oxide being present in amounts equal to 5 to 25 moles of
ethylene oxide per mole of alkyl phenol. The alkyl substituent in
such compounds may be derived from polymerized propylene,
diisobutylene, octene or nonene, for example.
2. Those nonionic synthetic detergents derived from the
condensation of ethylene oxide with the product resulting from the
reaction of propylene oxide and ethylene diamine. For example,
compounds containing from about 40 to about 80 percent
polyoxyethylene by weight and having a molecular weight of from
about 5,000 to 11,000 resulting from the reaction of ethylene oxide
groups with a hydrophobic base constituted of the reaction product
of ethylene diamine and excess propylene oxide, said base having a
molecular weight of the order of 2,500 to 3,000 are
satisfactory.
3. The condensation product of 1 mole of aliphatic alcohols having
from eight to 22 carbon atoms, in either straight chain or
branched-chain configuration, with from 5 to 40 moles of ethylene
oxide, e.g., a coconut alcohol-ethylene oxide condensate having
from 5 to 40 moles of ethylene oxide per mole of coconut alcohol,
the coconut alcohol fraction having from 10 to 14 carbon atoms.
4. The unsubstituted amides and the monoethanol and diethanol
amides of fatty acid having acyl moieties of from about eight to
about 22 carbon atoms. These acyl moieties are normally derived
from naturally occurring glycerides (e.g., coconut oil, palm oil,
soybean oil and tallow), but can be derived synthetically (e.g., by
the oxidation of petroleum, or by hydrogenation of carbon monoxide
by the Fischer-Tropsch process).
5. Long chain tertiary amine oxides corresponding to the following
general formula
wherein R.sup.1 is an alkyl radical of from about eight to about 22
carbon atoms, R.sup.2 and R.sup.3 are each methyl, ethyl or
hydroxyethyl radicals, R.sup.4 is ethylene, and n equals from zero
to about 10. The arrow in the formula is a conventional
representation of a semipolar bond. Specific examples of amine
oxide detergents include: dimethyldodecylamine oxide and
bis-(2-hydroxyethyl)-dodecylamine oxide.
6. Long chain tertiary phosphine oxides corresponding to the
following general formula RR'R"P O wherein R is an alkyl, alkenyl
or monohydroxyalkyl radical ranging from 10 to 22 carbon atoms in
chain length and R' and R" are each alkyl or monohydroxyalkyl
groups containing from one to three carbon atoms. The arrow in the
formula is a conventional representation of a semipolar bond.
Examples of suitable phosphine oxides are found in U.S. Pat. No.
3,304,263 which issued Feb. 14, 1967, and include:
dimethyldodecylphosphine oxide and
bis-(2-hydroxyethyl)dodecylphosphine oxide.
7. Long chain sulfoxides having the formula
wherein R.sup.5 is an alkyl radical containing from about 10 to
about 22 carbon atoms, from zero to about five ether linkages and
from zero to about two hydroxyl substituents, at least one moiety
of R.sup.5 being uninterrupted by ether linkages and containing
from about 10 to about 18 carbon atoms, and wherein R.sup.6 is an
alkyl radical containing from one to three carbon atoms and from
zero to two hydroxyl groups. Specific examples of these sulfoxides
are: dodecyl methyl sulfoxide and 3-hydroxy tridecyl methyl
sulfoxide.
The zwitterionic synthetic detergents suitable for use herein can
be broadly described as derivatives of aliphatic quaternary
ammonium, phosphonium and sulfonium compounds, in which the
aliphatic radical may be straight chain or branched, and wherein
one of the aliphatic substituents contains from about eight to 22
carbon atoms and one contains an anionic water solubilizing group,
e.g., carboxy, sulfo, sulfato, phosphate or phosphono. Examples of
compounds falling within this definition are
3-(N,N-dimethyl-N-hexadecylammonio) propane-1-sulfonate and
3-(N,N-diemthyl-N-hexadecylammonio)-2-hydroxy propane-1-sulfonate.
For more examples of zwitterionic synthetic detergents, see Diehl
and Smith, "Laundering Fabrics in Cold Water Containing a Synthetic
Detergent Composition," Canadian Pat. No. 708,147, issued Apr. 20,
1965 at pg. 6, lines 1- 22.
Mixtures of various nonionic detergents or mixtures of nonionic
detergents and zwitterionic detergents can be utilized to advantage
herein. Preferred herein are the condensation products of 1 mole of
aliphatic alcohol having eight to 22 carbon atoms with from 5 to 40
moles of ethylene oxide, e.g., tallow alcohol ethoxylated with 11
or 30 moles of ethylene oxide and coconut alcohol ethoxylated with
6 moles of ethylene oxide. Also preferred are the
3-(N,N-dimethyl-N-alkylammonio)-2-hydroxy propane-1-sulfonates
wherein the alkyl has from eight to 22 carbon atoms, e.g.,
3-(N,N-dimethyl-N-coconutalkylammonio)-2-hydroxy
propane-1-sulfonate. These compounds provide enhancement of
protease stability particularly at temperatures of up to about
80.degree. F. and provide excellent detergency and cleaning
properties to the compositions of the invention. In addition, they
provide desirable levels of .alpha.-amylase stabilization.
The stabilized compositions of the present invention are prepared
to contain from about 65 to about 97 percent by weight of water.
Preferably from about 72 to about 95 percent is employed.
Demineralized water is preferred, although not mandatory for use
herein.
The various components of the enzyme compositions of this invention
can be mixed together in any order. However, it is preferred that a
stabilizer-water mixture be prepared first and the enzymes added
thereto to prevent any degradation or deactivation in solutions
predominately consisting of either water or organic stabilizing
compound. The optional detergent components can be added at any
time.
The pH of the stabilized aqueous enzyme compositions of this
invention generally ranges from about 5.0 to 10.0 and preferably
ranges from about 6.5 to about 8.5. Maximum stabilizing affects are
obtained in the preferred pH range. The pH can be raised with a
base, e.g., sodium or potassium hydroxide, or lowered with an acid,
e.g., hydrochloric acid.
It is also preferred, although not mandatory, that a preservative
be added to the compositions to prevent bacterial and fungal
growth. Phenyl mercuric acetate which is generally utilized herein
in amounts ranging from about 10 to about 40 parts per million of
the compositions is an effective preservative. Any preservative
compatible with the components of the compositions can be utilized
herein.
The compositions of this invention can be employed as spot
removers, detergent additives or as a detergent cleaning
composition per se. These compositions can be packaged in
spray-type bottles and conveniently used to remove relatively small
spots from fabrics or can be employed in larger quantities as
additives to other detergent compositions. These compositions can
be substituted for hypochlorite bleaches as they remove many of the
stains which these bleaches remove, do not weaken textile fibers,
and do not attack or degrade fluorescers and whiteners. With the
addition of optional nonionic and/or zwitterionic detergents, these
compositions can be utilized per se as excellent cleaning
compositions under a variety of washing conditions.
EXAMPLES
The following examples merely serve to illustrate the invention in
specific detail and when read in conjunction with the foregoing
description will aid in determining the full scope of the present
invention. The examples are merely illustrative and are not
intended to restrict this invention. All parts, percentages and
ratios set forth herein are by weight unless otherwise
indicated.
The following compositions (examples 1 to 4) were prepared and
stored in closed glass bottles for the lengths of time indicated in
table I. Each composition contained 10 percent by weight of the
indicated organic stabilizing agent; 1 percent Alcalase
(proteolytic enzyme preparation having a crystalline enzyme content
of about 6 percent); and 89 percent of an aqueous stock solution
containing 0.01 percent calcium acetate monohydrate and 0.29
percent sodium chloride. The proteolytic activity of each
composition was measured at the stated intervals by the Azocoll
method. The Azocoll method is based on the release of a
water-soluble dye from a water-insoluble protein-dye substrate
(Azocoll) by a proteolytic enzyme. The amount of dye released under
carefully controlled conditions is measured spectrophotometrically.
Enzymatic activity is calculated from the amount of dye released.
Initial activity equals 100 percent.
Control samples stored under identical conditions were also
evaluated for retention of enzymatic activity. In Control-1, no
organic stabilizing agent was employed. In the case of Control-2,
no organic stabilizing agent was present and the stock solution was
replaced with distilled water, i.e., no calcium acetate monohydrate
or sodium chloride was present. The results are tabulated as
follows: ##SPC1##
The following stabilized aqueous enzyme compositions (examples 5 to
8) containing 1 percent of Monsanto CRD Protease (a commercially
available mixture of protease and amylases derived from Bacillus
subtilis) were prepared. Each sample contained sodium chloride,
calcium acetate monohydrate, organic stabilizer and water in the
amounts employed in examples 1 to 4. Control samples stored under
identical conditions containing no organic stabilizer (Control-1)
and no stabilizer, sodium chloride or calcium acetate (Control-2)
were also evaluated for enzyme stability. The Azocoll method
hereinbefore described was employed to measure the amount of
alkaline protease activity remaining after storage. One ml. of
0.002 M disodium ethylene-diaminetetraacetate was employed in each
Azocoll analysis to inhibit the activity of neutral protease. The
results are tabulated in table II as follows: ##SPC2##
The following stabilized compositions (examples 9 to 20) described
in table III were prepared. In each example, the water (containing
calcium acetate monohydrate and sodium chloride) and stabilizing
agent were thoroughly mixed, the nonionic or zwitterionic was added
and the enzyme added last. The enzymes were stabilized in each
example during high temperature (100.degree. F.) storage. All
perform well as spot removers, detergent additives and as
detergents per se. The nonionic and the zwitterionic additives
enhance storage stability, particularly at temperature of up to
about 80.degree. F., and provide excellent detergency properties
which make the compositions especially suited as spot removers,
detergent additives and as cleaning compositions per se. ##SPC3##
##SPC4##
The following table, table IV, describes additional aqueous
enzyme-containing compositions according to the invention having
enhanced enzyme stability and useful as spot removers, detergent
additives and as detergent compositions per se. ##SPC5##
EXAMPLE 39
A stabilized aqueous enzyme composition is formulated according to
this invention from the following components:
Component Weight % Alcalase (6% crystalline enzyme) 1% TAE.sub.30
5% Acetone 10% Water 84% Phenyl Mercuric Acetate 30 p.p.m.
The pH of this preparation is adjusted to 7.0 with sodium
hydroxide.
This composition can be employed without dilution as a soil- and
stain-removing composition even after extended periods of storage.
This composition sprayed directly onto stained or soiled areas of
textile materials facilitates the removal of a variety of
proteinaceous stains.
This composition can also be employed as an additive to commercial
detergent formulations. When about 1.2 ml. of the composition of
this invention is added per gallon of washing solution, excellent
soil- and stain-removing properties are observed.
EXAMPLE 40
Results substantially similar to those in the previous examples are
obtained when the following enzymes or commercial enzyme
compositions are substituted for Alcalase and Monsanto CRD Protease
(DA-10) in that the enzymes are stabilized in aqueous solutions:
Subtilisin, BPN', elastase, keratinase, carboxy peptidase, amino
peptidase, aspergillopeptidase A, aspergillopeptidase B,
collagenase, chymotrypsin, trypsin, pepsin, papain, bromelin,
Maxatase, Protease B-4000, Protease AP, Alcalase, CRD Protease,
Viokase, Pronase-P, Pronase-E, Pronase-AS, Pronase-AF, Bioprase,
Rapidase P-2000, Takamine, HI Proteolytic Enzyme 2000, Enzyme L-W,
Thozyme P-11 Concentrate, Pectinol, Rhozyme PF, Rhozyme J-25 and
Amprozyme 200.
Results substantially similar to those in the previous examples are
obtained when the following stabilizing agents are substituted for
those utilized in the previous examples in that the enzymes are
stabilized in aqueous solutions: ethylene glycol diethyl ether;
ethylene glycol di-n-propyl ether; ethylene glycol di-n-butyl
ether; diethylene glycol diethyl ether; triethylene glycol diethyl
ether; triethylene glycol di-n-butyl ether; the methyl ether of
pentaoxyethylenated butanol; the methyl ether of octaoxyethylenated
methanol; the butyl ether of decaoxyethylenated butanol; pyran;
1,3-dioxane; 1,4-dioxane; dioxolane; di-n-propyl ketone;
diisopropyl ketone; di-n-butyl ketone; and di-tert-butyl
ketone.
Results substantially similar to those in examples 9 through 38 are
obtained when the following nonionic and zwitterionic detergents
are substituted for the 3-(N,N-dimethyl-N-tallow
alkylammonio)propane-1-sulfonate, the
3-(N,N-dimethyl-N-middlecut-coconut-alkylammonio)-2-hydroxypropane1-sulfon
ate, dodecyldimethyl amine oxide and the coconut- and tallow-alkyl
ethoxylate compounds employed therein in that the stabilizing
effects of the organic stabilizing agents are enhanced: decyl
phenol ethoxylated with 20 moles of ethylene oxide per mole of
decyl phenol, hexadecanoic amide, hexadecanoic diethanol amide,
dimethyldodecylamine oxide, dimethyldodecylphosphine oxide and
dodecyl methyl sulfoxide, the condensation product of ethylene
oxide with the condensation product of propylene oxide with
propylene glycol, the ethylene oxide portion of the compound being
50 percent of the total weight of the compound being about 1,700;
the condensation product of ethylene oxide with the condensation
product of propylene oxide and ethylene diamine wherein the product
contains about 65 percent polyethylene oxide by weight and the
total molecular weight of the compound is 6,000.
Results substantially similar to those in examples 1 through 39 are
obtained when the pH of the compositions is maintained at 6.5, 8.0,
and 8.5, in that the enzymes are stabilized for long periods of
time. Enzyme stabilization is also obtained in examples 1 through
39 when the pH of the preparation is maintained at 5.0, 9.0, and
10.0.
The foregoing description of the invention has been presented
describing certain operable and preferred embodiments. It is not
intended that the invention should be so limited since variations
and modifications thereof will be obvious to those skilled in the
art, all of which are within the spirit and scope of this
invention.
* * * * *